Compositions and methods for array-based genomic nucleic acid analysis of biological molecules

ABSTRACT

The invention provides biological molecules modified by reaction with a compound having the formula: R 1 —X—R 2  , wherein R 1  is a cyclic ether group or an amino group, R 2  is an alkoxysilane group and X is a moiety chemically suitable for linking the cyclic ether group or the amino group to the alkoxysilane group. The invention also provides arrays, or “biochips,” comprising these modified biological molecules. Also provided are methods for making and using these compositions.

CROSS-REFERENCES TO RELATED APPLICATIONS

The present application is a Continuation-In-Part (CIP) of U.S. PatentApplications Serial No. (“U.S. Ser. No.”) 09/546,085, filed on Apr. 10,2000; which is a CIP of U.S. Ser. No. 09/071,876, filed on May 4, 1998,issued as U.S. Pat. No. 6,048,695, on Apr. 11, 2000. Theseaforementioned applications and patent are explicitly incorporatedherein by reference in their entirety and for all purposes.

TECHNICAL FIELD

The present invention claims a closely related family of compounds,devices, and methods relating to techniques for immobilizing biologicalmolecules, e.g., nucleic acids, to a solid support for the purpose ofconducting scientific investigation or routine testing upon the boundmolecule (e.g., nucleic acid) samples in areas such as genome-widegenetic mapping and gene expression studies, protein interactionstudies, peptide interaction studies and small molecule interactionswith larger macromolecules.

BACKGROUND

A large percentage of investigation in the biochemical arts is directedto studies involving nucleic acids, particularly deoxyribonucleic acid,or DNA. DNA is a water-soluble compound, that if left in solution (i.e.,a water-based solution), is likely to degrade, through hydrolysis, andso forth. Obviously this frustrates any investigation involving DNA, andso therefore, accurate and reliable study involving DNA requires amethod or device to ensure the integrity of DNA. To facilitate the studyof DNA, it is often desirable to affix or immobilize the DNA on a solidsurface, such as a smooth sheet of glass. Fixed in place in this manner,the DNA can be readily manipulated (i.e., reacted with othersubstances). If DNA is envisioned as a long strand, then immobilizingDNA means fixing one end of the strand to the solid support so that theremainder of the strand is unmodified and free to undergo furtherreaction depending upon the particular study. Indeed, this is a widelyused method to conduct laboratory studies involving DNA.

Perhaps the major problem associated with immobilizing DNA on a solidsupport is exactly how to do it without altering the DNA (other thanthat relatively small portion that is actually bound to the solidsupport). This is a very difficult problem because whatever solidsupport is used must be essentially inert. That is, it must not reactwith the DNA, other than simply to immobilize it upon the solid support.Glass is a particularly suitable solid support, because it isinexpensive, and highly inert. At present, the current orthodoxy is thatthe solid support (e.g., a glass surface) must first be primed orderivatized so that it can bind one end of the DNA to the surface.Numerous techniques exist to do this.

Unfortunately, derivatizing the otherwise inert surface of glass createsproblems that could confound the results of the laboratory studyinvolving DNA. One problem is that derivatizing the glass surfacecreates a net positive electrostatic charge on the glass surface. SinceDNA is (net) negatively charged, other DNA (or DNA used later in thestudy but not deliberately affixed to the glass surface) is prone tostick (by non-specific electrostatic attraction) to the glass surface.In other words, DNA “probes” which are single (rather than double)strands of DNA are often contacted with an array of DNA single strandsaffixed to a solid support. Since the probe has a known nucleotidesequence and since a particular single strand of DNA will bindpreferentially to a complementary strand, the particular immobilizedstrand to which the probe reacts reveals the nucleotide sequence of thepreviously unknown immobilized strand. Yet simple experiments of thistype (probe studies) are severely confounded by electrostatic stickingof the probe to the derivatized (hence electrostatically charged) glasssurface. For instance, the probe is often radiolabeled so that itspresence can be detected by an ordinary radiation detector. Thus, thelocation of the probe on the glass surface, as evidenced by thedetector, reveals the chemical identity or sequence of the immobilizedDNA strand at that particular location on the glass surface (which isknown and designated in advance). Yet the radiation detector is unableto distinguish between probe that is chemically bound to a complementarystrand of DNA affixed to the solid support, and probe that is simplyelectrostatically stuck to the glass surface (but not to a DNA strand).

Second, derivatized surfaces result in what shall be known as“spreading.” Spreading occurs because the solid support surface becomeshydrophilic upon derivatization. As a result, when the DNA (desired tobe immobilized upon the solid support) is contacted with the surface ofthe solid support, it spreads, rather than remaining in a discrete“spot,” which it should ideally do, since whether the radioactive probeis detected in one spot or another determines whether the scientistinfers that the probe reacted with this or that immobilized DNA.Spreading is a major constraint on array density (i.e., the number ofdifferent nucleic acid samples that can be arranged on a single solidsupport). Hence, any means to curtail spreading, and so increase arraydensity, is highly desirable.

One very common substance used to prepare a glass surface to receive anucleic acid sample is poly-L-lysine. See, e.g., DeRisi (1996) 14 NatureGenetics 457; Shalon (1996) 6 Genome Res. 639; and Schena (1995) 270Science 467. Other types of pre-derivatized glass supports arecommercially available (e.g., sialylated microscope slides). See, e.g.,Schena (1996) 93 Proc. Natl. Acad. Sci. USA 10614.

Numerous other surface coatings have been disclosed. See, e.g., U.S.Pat. No. 5,630,932, discloses a coating for a probe (platinum) tip foruse in scanning tunneling microscopy; numerous means are disclosed forcoating the surface, notably, Si(OCH₃)CH₂I. U.S. Pat. No. 5,610,287,discloses coating a solid support with a salt or cationic detergent tonon-covalently bond nucleic acids to the support. U.S. Pat. No.5,024,933, discloses coating a solid support with an isolate ofnaturally occurring mussel adhesive protein. U.S. Pat. No. 4,937,188,discloses covalently bonding an enzyme to a solid support via molecularchain which acts as a substrate for the enzyme. U.S. Pat. No. 4,818,681,discloses coating a solid support with a nucleoside phosphate throughthe heterocyclic moiety of the nucleoside; the nucleic acid is thenimmobilized upon the solid support by enzymatic coupling. U.S. Pat. No.4,806,631, discloses activating a nylon solid support by partiallysolvolyzing the amine groups (e.g., by treating with an alkylatinggroup) on the nylon surface.

Another approach to this problem involves derivatizing both the solidsupport and the nucleic acid sought to be immobilized. See, e.g., U.S.Pat. No. 5,641,630, discloses coating a solid support with a complexingagent that binds to another complexing agent to which the nucleic acidsought to be bound is likewise bound. U.S. Pat. No. 5,554,744, disclosescontacting a solid support with diisopropylcarbodiimide and an acidcatalyst and a succinylated nucleoside to immobilize the nucleoside.U.S. Pat. No. 5,514,785, discloses coating a solid support with,preferably, primary and secondary amines, followed by activation of thenucleic acid using cyanuric chloride. U.S. Pat. No. 5,215,882, disclosesmodifying the nucleic acid sought to be immobilized with a primary amineor equivalent, followed by reaction of the modified nucleic acid withthe solid support (the support must have free aldehyde groups) in thepresence of a reducing agent.

Finally, a third approach to the problem of immobilizing nucleic acidsto solid support material involves creating a novel solid support. See,e.g., U.S. Pat. Nos. 5,055,429, 5,008,220, 4,963,436, 4,826,790, and4,826,789, disclose solid support material made from aluminosilicatematerial.

Due to the aforementioned shortcomings of derivatizing the (entire)glass surface prior to affixing the nucleic acid samples, severalmethods have been developed which involve synthesizing the nucleic acidsamples directly to the solid support. See, e.g., Hacia (1996) 14 NatureGenetics 441 (1996); Lockhart (1996) 14 Nature Biotechnology 1675(1996); Maskos (1992) 20 Nucleic Acids Res. 1679 (1992).

To reiterate: at present, the prevailing view in the biochemical arts isthat, in order to effectively immobilize nucleic acids onto solidsurfaces, the solid support must first be derivatized, or madechemically labile, so that the nucleic acid can then be reacted withsolid support. In addition, epoxides are known mutagens; that is, theyare known to damage nucleic acids, particularly DNA.

SUMMARY

This invention provides compositions and methods for affixing biologicalmolecules to solid supports. It demonstrates that any biologicalmolecule can be modified and affixed to an unmodified solid support. Askilled artisan will recognize the significance of first modifying amolecule to enhance its binding affinity by appropriate modifications;thus, this modified molecule can be immobilized to an unmodified solidsurface to generate a fully functional array of molecules for a spectrumof specific applications.

In one aspect, the invention provides any biological molecule, e.g., DNAand nucleic acids more generally, that are modified such that theyreadily adhere to an unmodified or underivatized glass surface. Inparticular, in one aspect of the invention epoxide-modified nucleicacids, particularly DNA, are readily affixed to an unmodified solidsupport.

The invention provides a modified biological molecule comprising abiological molecule modified by reaction with a compound having theformula:R₁—X—R₂,wherein R₁ is a cyclic ether group or an amino group, R₂ is analkoxysilane group and X is a moiety chemically suitable for linking thecyclic ether group or the amino group to the alkoxysilane group. In oneaspect, the R₁ cyclic ether is a compound comprising an epoxide groupsuch as an ethylene oxide, or equivalent. In alternative aspects, thecyclic ether is an oxirane group, or equivalent, or a compoundcomprising an aromatic hydrocarbon epoxide group.

In one aspect, the R₁ group reacts with the biological molecule suchthat the modified biological molecule is linked to the compound throughR₁ group. The linkage, or association, of the R₁ group to the biologicalmolecule can be such that the R₁ group is covalently or non-covalentlybound to the biological molecule.

In alternative aspects, the biological molecule comprises a nucleic acid(e.g., a oligonucleotide), a lipid, a polysaccharide, a polypeptide(e.g., a peptide), or an analog or a mimetic thereof, or a combinationthereof. The nucleic acid can comprise a DNA (e.g., a genomic DNA or acDNA), an RNA (e.g., an mRNA, rRNA, and the like) or an analog or amimetic thereof or a combination thereof. The nucleic acid can furthercomprise a telomeric structure or a chromatin structure.

In one aspect, the nucleic acid is attached to the compound by the R₁group, i.e., the nucleic acid reacts with the R₁ group at its 5′ end.

In one aspect, the cyclic ether is an epoxide group and the alkoxysilaneis —Si(OCH₃)₃, —Si(OC₂H₅)₃, —Si(OCH₃)H₂, —Si(OCH₃)(CH₃)_(2,) or—Si(OCH)₃)₂CH₃. In one aspect, the cyclic ether is an epoxide group andthe compound is 3-glycidoxypropyltrimethoxysilane.

In one aspect, the R₁ group is a primary amino group. In one aspect, theR₁ group is an amino group and the alkoxysilane is selected from thegroup consisting of —Si(OCH₃)₃, —Si(OC₂H₅)₃ and

-   -   wherein R₁, R₂ and R₃ are selected from the group consisting of        —H, —CH₃, —OCH₃, and —OC₂H₃, and provided that at least one of        R₁, R₂ or R₃ is either —OCH₃ or —OC₂H₃.

In one aspect, the R₁ group is an amino group and the compound is3-aminopropyltriethoxysilane.

The invention provides an article of manufacture comprising an arrayedplurality of biological molecules covalently bound to a surface,wherein, before attachment to the surface, the biological molecules aremodified by reaction with a compound having the formula: R₁—X—R₂ ,wherein R₁ is a cyclic ether group or an amino group, R₂ is analkoxysilane group and X is a moiety chemically suitable for linking thecyclic ether group or the amino group to the alkoxysilane group, andupon attachment to the surface the modified biological molecules arecovalently bound to the surface; wherein each biological molecule isattached to the surface on at least one discrete and known location toform a cluster of substantially identical biological molecules. Inalternative aspects of the article of manufacture the surface is aglass, a mica, a quartz, or a metal oxide surface. The metal oxidesurface can be an alumina (Al₂O₃), a titania (TiO₂), a SnO₂, a RuO₂, ora PtO₂, or an equivalent thereof. The surface of the article ofmanufacture can comprise a polystyrene, a polyester, a polycarbonate, apolyethylene, a polypropylene or a nylon.

In one aspect, the modified biological molecules are covalently bound tothe surface via the R₂ group.

On one aspect of the article of manufacture, the biological moleculescan comprise a nucleic acid, a lipid, a polypeptide, a polysaccharide,or an analog or a mimetic thereof, or a combination thereof. Inalternative aspects, the biological molecules are derived from a virus,a bacteria, a yeast, a plant, an insect, a mammal, such as a human or amouse. The biological molecules can comprise nucleic acids or analogs ormimetics thereof. The nucleic acids can comprise DNA, RNA or analogs ormimetics thereof or a combination thereof. The nucleic acids can beoligonucleotides.

In one aspect of the article of manufacture, the nucleic acids reactwith the R₁ group at their 5′ end.

In one aspect, the nucleic acids immobilized on the article ofmanufacture can comprise a plurality of fragments of a genomic nucleicacid. The biological molecule, e.g., a genomic nucleic acid or RNA, canbe derived from a normal cell or an abnormal cell, such as a cellsuspected of having a chromosomal defect or abnormality, e.g., a canceror tumor cell. In alternative aspects, the genomic DNA is derived from avirus, a bacteria, a yeast, a plant, an insect, a mammal, such as ahuman or a mouse.

In one aspect of the article of manufacture, the fragments of nucleicacid, e.g., genomic nucleic acid, further comprise a cloning vehicle.The cloning vehicle can comprise a bacterial artificial chromosome(BAC). In alternative aspects, the cloning vehicle comprises a plasmid,a cosmid, a bacteriophage P1-derived vector (PAC), a yeast artificialchromosome (YAC) or a mammalian artificial chromosome (MAC).

In one aspect of the article of manufacture, the nucleic acid comprisesa plurality of CpG island tags.

In one aspect of the article of manufacture, the fragments of genomicnucleic acid comprise sequences representing at least one substantiallycomplete chromosome or at least one defined section of a chromosome. Inone aspect, each genomic nucleic acid fragment has been mapped to aknown location on a chromosome. In alternative aspects, the nucleicacid, e.g., the genomic nucleic acid fragments, have a size no more thanabout 1.5 megabase, no more than about 1.2 megabase, no more than about1.0 megabase, and, no more than about 0.75 megabase in size.

In alternative aspects of the article of manufacture, each cluster ofsubstantially identical biological molecules consists of between about 5and about 400, or, between about 10 and about 200, or, between about 50and 100, substantially identical copies of a biological molecule. Thesurface can consist of less than about 800, about 600, about 500, about400, about 300, about 200 or about 100 clusters per square centimeter.

In alternative aspects of the article of manufacture, each cluster ofsubstantially identical biological molecules is about 100 microns, about50 microns, about 25 microns, about 15 microns or about 10 microns indiameter or smaller.

The invention provides an article of manufacture (e.g., array orbiochip) comprising an array of cloned genomic nucleic acid fragmentsrepresenting a defined subsection of or a substantially completechromosome, wherein, before attachment to the surface, the clonedfragments are modified by reaction with a compound having the formula:R₁—X—R₂, wherein R₁ is an epoxide group, R₂ is an alkoxysilane group andX is a moiety chemically suitable for linking the epoxide group and thealkoxysilane group, and the modified cloned fragments are covalentlybound to the surface; wherein each array-bound cloned fragment has beenmapped to a known location on a chromosome.

The invention provides a kit comprising an article of manufacture of theinvention, as described herein, and printed matter, wherein the printedmatter comprises instructions on hybridizing a sample of nucleic acid toan array-bound nucleic acid.

The invention provides a method for identifying a specific bindingpartner, comprising: (a) providing an article of manufacture comprisingan arrayed plurality of biological molecules covalently bound to asurface, wherein, before attachment to the surface, the biologicalmolecules are modified by reaction with a compound having the formula:R₁—X—R₂, wherein R₁ is an cyclic ether group or an amino group, R₂ is analkoxysilane group and X is a moiety chemically suitable for linking thecyclic ether group or the amino group to the alkoxysilane group, andupon attachment the modified biological molecules are covalently boundto the surface, wherein each biological molecule is attached to thesurface on at least one discrete and known location to form a cluster ofidentical biological molecules; (b) providing a sample of biologicalmolecules; (c) contacting the sample of step (b) with the array-boundbiological molecules as set forth in step (a) under conditionspermissive for specific binding of a molecule in the sample of step (b)to an array-bound biological molecule; and, (d) screening for specificbinding of a molecule in the sample of step (b) to an array-boundbiological molecule, thereby identifying a specific binding partner. Inone aspect, the method further comprises at least one wash step betweenthe contacting of step (c) and the screening of step (d).

The invention provides a method for generating a molecular profile of anucleic acid sample, comprising the following steps: (a) providing anarticle of manufacture comprising an array of biological molecules,wherein, before attachment to the surface, the biological molecules aremodified by reaction with a compound having the formula: R₁—X—R₂,wherein R₁ is a cyclic ether group, R₂ is an alkoxysilane group and X isa moiety chemically suitable for linking the cyclic ether group and thealkoxysilane group, and the modified biological molecules are covalentlybound to the surface; (b) providing a sample comprising a nucleic acid;and (c) contacting the nucleic acid with the array-bound biologicalmolecules as set forth in step (a) under conditions, allowing binding ofthe sample nucleic acid to the array-bound biological molecules, anddetecting binding of the sample nucleic acid to the array-boundbiological molecules, thereby generating a molecular profile of thesample nucleic acid.

In one aspect of this method, the array-bound biological moleculescomprise a nucleic acid, such as a DNA corresponding to, or derivedfrom, a genomic DNA, or, a message. The binding can comprisehybridization of the sample nucleic acid to the array-bound nucleicacid. The array-bound nucleic acid can represent a section of at leastone a chromosome or at least one substantially complete chromosome. Thechromosome can be a viral, a bacterial, a yeast, a plant, an insect, ora mammalian, such as a human or a mouse, chromosome. In one aspect, thearray-bound nucleic acids have been mapped to a known location on achromosome.

In one aspect of this method, the molecular profile is a comparativegenomic hybridization (CGH). The molecular profile can comprisedetection of a genomic DNA amplification, a genomic DNA deletion, or agenomic DNA insertion. The molecular profile can comprise detection of apoint mutation.

In one aspect of this method, the molecular profile is theidentification of a single or multiple point mutations, such as asingle-nucleotide polymorphism (SNP). In one aspect of this method, thedetection of a point mutation can further comprise use of a primerextension assay.

In one aspect of this method, the modified nucleic acid of theinvention, or the array-bound nucleic acids, comprise on or more CpGisland tags. In one aspect, the molecular profile is generated by adifferential methylation hybridization (DMH) reaction. The samplenucleic acids can comprise genomic DNA digested with at least onemethylation-sensitive restriction endonuclease and the molecular profilecomprises detection and mapping of hypermethylated regions of thegenome. The methylation-sensitive restriction endonuclease can beselected from the group consisting of NotI, SmaI, SacII, EagI, MspI,HpaII and BssHII.

In one aspect of this method, the molecular profile comprises detectionof transcriptionally active regions of a genome. In one aspect, thesample of nucleic acid can be derived from a nuclear run-off assay; thissample can be modified by the methods of the invention, and, in oneaspect, these modified nucleic acids are immobilized onto an array asset forth in the invention.

In one aspect of this method, the molecular profile comprises ananalysis of a chromatin structure. The modified nucleic acids of theinvention, or the array-bound biological molecule, can comprise achromatin structure.

In one aspect of this method, the molecular profile comprises ananalysis of a telomeric structure. The molecular profile of a telomericstructure can comprise an analysis of telomeric erosion or telomericaddition. The modified nucleic acids of the invention, or thearray-bound biological molecule (e.g., nucleic acid), can comprise oneor more telomere structures.

The invention provides a method for making a modified biologicalmolecule comprising (a) providing a biological molecule; (b) providing acompound having the formula: R₁—X—R₂, wherein R₁ is a cyclic ether groupor an amino group, R₂ is an alkoxysilane group and X is a moietychemically suitable for linking the cyclic ether group or the aminogroup to the alkoxysilane group; and (c) reacting the biologicalmolecule with the compound, thereby modifying the biological moleculewith the compound.

The invention provides a method for making an article of manufacture(e.g., array or biochip) comprising an arrayed plurality of biologicalmolecules covalently bound to a surface comprising (a) providing abiological molecule; (b) providing a compound having the formula:R₁—X—R₂, wherein R₁ is a cyclic ether group or an amino group, R₂ is analkoxysilane group and X is a moiety chemically suitable for linking thecyclic ether group or the amino group to the alkoxysilane group; (c)providing a surface comprising hydroxyl groups; (d) reacting thebiological molecule with the compound, thereby modifying the biologicalmolecule with the compound; and, (e) depositing a plurality of modifiedbiological molecules on the surface as discrete clusters, wherein amodified biological molecule is attached to the surface on at least onediscrete and known location to form at least one cluster ofsubstantially identical biological molecules; the array comprises atleast two, a plurality of, clusters.

In one aspect of the method for making an article of manufacture, thecompound used to modify the biological molecule (e.g., a nucleic acid)is a 3-glycidoxypropyltrimethoxysilane. In one aspect, the biologicalmolecule is a nucleic acid and the reaction with the3-glycidoxypropyltrimethoxysilane is at a basic pH, thereby generating amodified nucleic acid. In this reaction, the pH can be above about pH9.5. A 3-glycidoxypropyltrimethoxysilane modified nucleic acid can bedeposited on an underivatized glass surface at about a neutral pH.

In another aspect of the method for making an article of manufacture,the compound used to modify the biological molecule (e.g., a nucleicacid) is a 3-aminopropyltriethoxysilane. In one aspect, the biologicalmolecule is a nucleic acid and the reaction with the3-aminopropyltriethoxysilane is at about a neutral pH. This reaction cantake place in the presence of sodium bisulfate, or equivalent. Thismodified nucleic acid can be deposited on an underivatized glasssurface.

The modified biological molecules, such as a modified nucleic acid, ofthe invention, will adhere to a solid surface to allow subsequentbiochemical investigations. Thus, in one aspect of the presentinvention, a modified biological molecule, such as a modified nucleicacid, comprises a biological molecule (e.g., a nucleic acid) covalentlybound to moiety containing two crucial functional groups: a cyclic ethergroup and an alkoxysilane group. In accordance with other aspects of thepresent invention, methods for preparing the aforementioned modifiedbiological molecules (e.g., nucleic acids) are claimed.

In another aspect, the invention provides a high-density microarraycomprising a glass or other inert surface. This array, or “biochip,” canbe made by printing numerous highly discrete modified biologicalmolecule (e.g., DNA) sample spots, or “clusters,” upon the surface.

In another aspect, the invention provides a modified biological molecule(e.g., a nucleic acid) prepared from a biological molecule (e.g., anucleic acid) and a halogenated silane, or equivalent.

In another aspect, the invention provides a modified nucleic acidprepared by reaction of a biological molecule (e.g., a nucleic acid)with a brominated moiety, followed by reaction with an aminated silane.

In another aspect, the invention provides a device that allows printingof the aforementioned high-density microarrays.

In another aspect, the invention provides modified silanes that allowthe skilled artisan to modulate the electrostatic properties of thesolid surface to optimize sample density and detection sensitivity.

The present invention possesses numerous advantages over the prior art.Many of the advantages derive from the fact that the solid surface,which is can be ordinary glass, remains highly chemically inert. Thus,the previously mentioned problems of probe (or other reactant) stickingto the glass as well as “spreading” are entirely eliminated. Theultimate result is, among other things, far higher detection sensitivitycompared with state-of-the-art derivatized solid support.

In addition, the biological molecule (e.g., a nucleic acid) to beimmobilized upon the solid support is readily derivatized. The reactionof the epoxide derivatives of the invention is simple to execute; itoccurs under mild conditions, reaction rates are quick, and equilibriumis highly favorable. Moreover, the epoxide-modified biological molecules(e.g., nucleic acids) of the present invention are essentiallypermanently stable; thus they can be prepared (derivatized) and storedfor later use (reaction with a non-derivatized surface).

The details of one or more aspects of the invention are set forth in theaccompanying drawings and the description below. Other features,objects, and advantages of the invention will be apparent from thedescription and drawings, and from the claims.

All publications, GenBank Accession references (sequences), ATCCDeposits, patents and patent applications cited herein are herebyexpressly incorporated by reference for all purposes.

DESCRIPTION OF DRAWINGS

FIG. 1 depicts a coupling reaction of nucleic acid (in this instanceDNA) with 3-glycidoxypropyltrimethoxysilane, followed by the reaction ofthe newly modified DNA and the solid support (in this instance a glasssurface). The final reaction product, the immobilized DNA, is shown atbottom.

FIG. 2 depicts a coupling reaction of nucleic acid (in this instanceDNA) with 3-aminoproplytriethoxysilane followed by the reaction of thenewly modified DNA and the solid support (in this instance a glasssurface). The final reaction product, the immobilized DNA, is shown atbottom.

FIG. 3 depicts a device for making a high-density microarray; both a top(FIG. 3A) and a side view (FIG. 3B) are shown.

FIG. 4 depicts the silanization of nucleic acid through alkylation ofhalogen-containing silane compounds.

FIG. 5 a depicts the first step in the silanization of nucleic acidusing amine-containing silane compounds. In this case, the reactionoccurs preferentially at the guanine base at neutral and slightly basicpH.

FIG. 5 b depicts the first step in the silanization of nucleic acidusing amine-containing silane compounds. In this case, the reactionoccurs preferentially at the cytosine base at more basic pH.

FIG. 5 c depicts the second and final step in the silanization ofnucleic acid using amine-containing silane compounds.

FIG. 6 is a schematic representation of one aspect of the presentinvention showing silane linkers by hydrophobic linkers.

FIG. 7 is a schematic representation of an exemplary reaction wherein abiological molecule, a polypeptide, is modified, or “activated,” by amethod comprising use of succinimidyl acetylthiopropionate (SATP) tointroduce an active sulfhydryl functional group, as described in detailin Example 9, below.

Drawings are not necessary to scale. Certain features of the inventionmay be exaggerated in scale or shown in schematic form in the interestof clarity and conciseness. Like reference symbols in the variousdrawings indicate like elements.

DETAILED DESCRIPTION

The invention provides modified biological molecules, such aspolypeptides and nucleic acids, and articles of manufacture comprisingarrays, with these modified biological molecules immobilized to thearray surface. The invention also provides methods for making and usingthese compositions.

One aspect of the invention is chemical modification of the biologicalmolecule (e.g., nucleic acid) sought to be immobilized. This chemicallymodified nucleic acid is then readily reacted to a solid support such asa glass surface, rendering the biological molecule (e.g., nucleic acid)immobilized. Again, this is in direct contradiction to the prior art,which teaches modification of the solid support, rather than the nucleicacid itself.

The modified the biological molecules (e.g., nucleic acids) of thepresent invention readily adhere to a variety of solid surfaces havingreactive functional groups, e.g., hydroxyl groups. These include, thoughare not limited to: quartz glass, mica, alumina (Al₂O₃), titania (TiO₂),SnO₂, RuO₂, PtO₂, plastics such as the following polymer materials,polystyrene, polyester, polycarbonate, polyethylene, polypropylene, andnylon as well as numerous semi-conductive surfaces, such as numerousother metal oxide surfaces and equivalents.

In one family of aspects, the chemically modified biological molecules(e.g., nucleic acids) of the present invention are so modified withcompounds having two crucial functionalities: a ring ether and analkoxysilane group. The biological molecule (e.g., nucleic acid) reactswith the ring ether, then the newly modified biological molecules (e.g.,nucleic acids) are contacted with the otherwise inert surface (e.g.,glass), where the alkoxysilane group reacts with a hydroxyl-containing(e.g., hydroxyl derivatized) surface, e.g., Si—OH groups on the glasssurface.

In another distinct family of aspects, the chemically modifiedbiological molecules (e.g., nucleic acids) of the present invention areso modified with compounds having two crucial functionalities: an aminogroup and an alkoxysilane group. The biological molecules (e.g., nucleicacid) react with the amino group, then the newly modified biologicalmolecules (e.g., nucleic acids) are contacted with the otherwise inert(e.g., glass) surface, where the alkoxysilane group reacts with ahydroxyl-containing (e.g., hydroxyl derivatized) surface, e.g., Si—OHgroups on the glass surface.

In yet another distinct family of aspects, the biological molecules(e.g., nucleic acids) are modified by reaction with halogenated silanecompounds.

In another set of aspects, the biological molecules (e.g., nucleicacids) are derivatized by a two-step process involving a final reactionwith amine-containing silanes and brominated nucleic acids.

Other aspects are directed to preparing and optimizing high-densitymicroarrays utilizing the modified biological molecules (e.g., nucleicacids) of the other aspects of the present invention.

Further aspects include compositions and methods of making and usingthat comprise any biological molecule or combinations of biologicalmolecules. One skilled in the art realizes that nucleic acids, e.g., DNAor RNA, are only one of many biological compositions, e.g., biologicalpolymers, that can be modified by the methods of the invention and usedin the methods of the invention. A polymer refers to a molecule that hasjoined prefabricated units, e.g., monomers or compositions that can beof limited diversity, linked together, usually by identical mechanisms,e.g., a cellulose is a polymer is simple sugars or polysaccharides.Exemplary biological molecules include but are not limited to DNA, RNA,protein, peptides, lipids, saccharides, polysaccharides and mimetics andanalogs thereof. Thus, a skilled artisan recognizes that any biologicalmolecule, including those having a structure found in nature or asynthetic structure, including polymers, can be modified by the methodsof the invention and affixed to a solid surface similar to the modifiednucleic acids of the invention.

Another aspect of the invention is the modification of biologicalmolecules. One type of modification is chemical cross-linking. It iswell known in the art that bifunctional “crosslinking” reagents containtwo reactive groups, thus providing a means of covalently crosslinkingtwo target groups. The reactive groups in a chemical crosslinkingreagent typically belong to the classes of functional groups, e.g.,succinimidyl esters, maleimides and iodoacetamides. Bifunctionalcrosslinking reagents can be divided in homobifunctional,heterobifunctional and zero-length bifunctional crosslinking reagents.In homobifunctional crosslinking reagents, the reactive groups areidentical. These reagents couple like functional groups, e.g., twothiols, two amines, two acids or two alcohols, and are predominantlyused to form intramolecular crosslinks. In heterobifunctionalcrosslinking reagents, the reactive groups have dissimilar chemistry,allowing the formation of crosslinks between unlike functional groups.The “zero-length” crosslinking reagent forms a chemical bond between twogroups without itself being incorporated into the product. For example,water-soluble cardodiimide (EDAC) is used to couple carboxylic acids toamines.

In addition to the traditional bifunctional crosslinking reagents, anoncovalent interaction between two molecules that has very slowdissociation kinetics can also function as a crosslink. For example,reactive derivatives of phospholipids can be used to link the liposomesor cell membranes to antibodies or enzymes. Biotinylation andhaptenylation reagents can also be thought of as heterobifunctionalcrosslinking reagents because they comprise a chemically reactive groupas well as a biotin or a hapten moiety that binds with high affinity toavidin or an anti-hapten antibody, respectively.

In contrast to chemical crosslinking reagents, photoreactivecrosslinking reagents are available. The general scheme involvesphotoreactive crosslinking reagents that contain a chemically reactivegroup as well as a photoreactive group. These crosslinkers are firstchemically reacted with one molecule and then this modified molecule iscoupled to a second molecule using UV illumination. Depending on thereactive properties of the chemical and photoreactive groups, thesecrosslinkers can be used to couple like or unlike functional groups.

Other aspects are directed to preparing and optimizing high-densitymicroarrays utilizing the modified molecules of the invention.

Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the meaning commonly understood by a person skilled in the art towhich this invention belongs. As used herein, the following terms havethe meanings ascribed to them unless specified otherwise.

The term “nucleic acid” as used herein refers to a deoxyribonucleotide(DNA) or ribonucleotide (RNA) in either single- or double-stranded form.The term encompasses nucleic acids containing known analogues of naturalnucleotides. The term encompasses mixed oligonucleotides comprising anRNA portion bearing 2′-O-alkyl substituents conjugated to a DNA portionvia a phosphodiester linkage, see, e.g., U.S. Pat. No. 5,013,830. Theterm also encompasses nucleic-acid-like structures with syntheticbackbones. DNA backbone analogues provided by the invention includephosphodiester, phosphorothioate, phosphorodithioate, methylphosphonate,phosphoramidate, alkyl phosphotriester, sulfamate, 3′-thioacetal,methylene(methylimino), 3′-N-carbamate, morpholino carbamate, andpeptide nucleic acids (PNAs); see Oligonucleotides and Analogues, aPractical Approach, edited by F. Eckstein, IRL Press at OxfordUniversity Press (1991); Antisense Strategies, Annals of the New YorkAcademy of Sciences, Volume 600, Eds. Baserga and Denhardt (NYAS 1992);Milligan (1993) J. Med. Chem. 36:1923-1937; Antisense Research andApplications (1993, CRC Press). PNAs contain non-ionic backbones, suchas N-(2-aminoethyl) glycine units. Phosphorothioate linkages aredescribed, e.g., by U.S. Pat. Nos. 6,031,092; 6,001,982; 5,684,148; seealso, WO 97/03211; WO 96/39154; Mata (1997) Toxicol. Appl. Pharmacol.144:189-197. Other synthetic backbones encompassed by the term includemethyl-phosphonate linkages or alternating methylphosphonate andphosphodiester linkages (see, e.g., U.S. Pat. No. 5,962,674;Strauss-Soukup (1997) Biochemistry 36:8692-8698), and benzylphosphonatelinkages (see, e.g., U.S. Pat. No. 5,532,226; Samstag (1996) AntisenseNucleic Acid Drug Dev 6:153-156). The term nucleic acid is usedinterchangeably with gene, DNA, RNA, cDNA, mRNA, oligonucleotide primer,probe and amplification product.

The terms “polypeptide,” “protein,” and “peptide” include compositionsof the invention that also include “analogs,” or “conservative variants”and “mimetics” or “peptidomimetics” with structures and activity thatsubstantially correspond to the polypeptide from which the variant wasderived, as discussed in detail, below.

The term “small molecule” means any synthetic small molecule, such as anorganic molecule or a synthetic molecule, such as those generated bycombinatorial chemistry methodologies. These small molecules can besynthesized using a variety of procedures and methodologies, which arewell described in the scientific and patent literature, e.g., OrganicSyntheses Collective Volumes, Gilman et al (Eds) John Wiley & Sons,Inc., NY; Venuti (1989) Pharm Res. 6:867-873. Synthesis of smallmolecules, as with all other procedures associated with this invention,can be practiced in conjunction with any method or protocol known in theart. For example, preparation and screening of combinatorial chemicallibraries are well known, see, e.g., U.S. Pat. Nos. 6,096,496;6,075,166; 6,054,047; 6,004,617; 5,985,356; 5,980,839; 5,917,185;5,767,238.

The terms “array” or “microarray” or “biochip” or “chip” as used hereinis an article of manufacture, a device, comprising a plurality ofimmobilized target elements, each target element comprising a “cluster”or “biosite” or defined area comprising a biological molecule (e.g., anucleic acid molecule or polypeptide, such as an antibody) immobilizedto a solid surface, as discussed in further detail, below.

The term “sample of nucleic acid targets” or “sample of nucleic acid” asused herein refers to a sample comprising DNA or RNA, or nucleic acidrepresentative of DNA or RNA isolated from a natural source, in a formsuitable for hybridization (e.g., as a soluble aqueous solution) toanother nucleic acid or polypeptide or combination thereof (e.g.,immobilized probes). The nucleic acid may be isolated, cloned oramplified; it may be, e.g., genomic DNA, mRNA, or cDNA fromsubstantially an entire genome, substantially all or part of aparticular chromosome, or selected sequences (e.g. particular promoters,genes, amplification or restriction fragments, cDNA, etc.). The nucleicacid sample may be extracted from particular cells or tissues. The cellor tissue sample from which the nucleic acid sample is prepared istypically taken from a patient suspected of having a genetic defect or agenetically-linked pathology or condition, e.g., a cancer, associatedwith genomic nucleic acid base substitutions, amplifications, deletionsand/or translocations. Methods of isolating cell and tissue samples arewell known to those of skill in the art and include, but are not limitedto, aspirations, tissue sections, needle biopsies, and the like.Frequently the sample will be a “clinical sample” which is a samplederived from a patient, including sections of tissues such as frozensections or paraffin sections taken for histological purposes. Thesample can also be derived from supernatants (of cells) or the cellsthemselves from cell cultures, cells from tissue culture and other mediain which it may be desirable to detect chromosomal abnormalities ordetermine amplicon copy number. In some cases, the nucleic acids may beamplified using standard techniques such as PCR, prior to thehybridization. In alternative aspects, the target nucleic acid may beunlabeled, or labeled (as, e.g., described herein) so that its bindingto the probe (e.g., oligonucleotide, or clone, immobilized on an array)can be detected. The probe an be produced from and collectively can berepresentative of a source of nucleic acids from one or more particular(pre-selected) portions of, e.g., a collection of polymerase chainreaction (PCR) amplification products, substantially an entirechromosome or a chromosome fragment, or substantially an entire genome,e.g., as a collection of clones, e.g., BACs, PACs, YACs, and the like(see below). The probe or genomic nucleic acid sample may be processedin some manner, e.g., by blocking or removal of repetitive nucleic acidsor by enrichment with selected nucleic acids.

Generating and Manipulating Nucleic Acids

The invention provides modified nucleic acids, and articles ofmanufacture comprising arrays that include modified nucleic acidcompositions and methods for making and using these arrays. The nucleicacid is modified by reaction with a compound having the formula:R₁—X—R₂, where R₁ is a cyclic ether group or an amino group, R₂ is analkoxysilane group and X is a moiety chemically suitable for linking thecyclic ether group or the amino group to the alkoxysilane group. Themodified nucleic acid or the immobilized nucleic acid on the array canbe representative of genomic DNA, including defined parts of, or entire,chromosomes, or entire genomes. In several aspects, the arrays andmethods of the invention are used in comparative genomic hybridization(CGH) reactions, including CGH reactions on arrays (see, e.g., U.S. Pat.Nos. 5,830,645; 5,976,790), see discussion below. The invention can bepracticed in conjunction with any method or protocol or device known inthe art, which are well described in the scientific and patentliterature.

General Techniques

The nucleic acids used to practice this invention, whether RNA, cDNA,genomic DNA, vectors, viruses or hybrids thereof, may be isolated from avariety of sources, genetically engineered, amplified, and/orexpressed/generated recombinantly (recombinant polypeptides can bemodified or immobilized to arrays in accordance with the invention). Anyrecombinant expression system can be used, including bacterial,mammalian, yeast, insect or plant cell expression systems.

Alternatively, these nucleic acids can be synthesized in vitro bywell-known chemical synthesis techniques, as described in, e.g.,Carruthers (1982) Cold Spring Harbor Symp. Quant. Biol. 47:411-418;Adams (1983) J. Am. Chem. Soc. 105:661; Belousov (1997) Nucleic AcidsRes. 25:3440-3444; Frenkel (1995) Free Radic. Biol. Med. 19:373-380;Blommers (1994) Biochemistry 33:7886-7896; Narang (1979) Meth. Enzymol.68:90; Brown (1979) Meth. Enzymol. 68:109; Beaucage (1981) Tetra. Lett.22:1859; U.S. Pat. No. 4,458,066. Double stranded DNA fragments may thenbe obtained either by synthesizing the complementary strand andannealing the strands together under appropriate conditions, or byadding the complementary strand using DNA polymerase with a primersequence.

Techniques for the manipulation of nucleic acids, such as, e.g.,subcloning, labeling probes (e.g., random-primer labeling using Klenowpolymerase, nick translation, amplification), sequencing, hybridizationand the like are well described in the scientific and patent literature,see, e.g., Sambrook, ed., MOLECULAR CLONING: A LABORATORY MANUAL (2NDED.), Vols. 1-3, Cold Spring Harbor Laboratory, (1989); CURRENTPROTOCOLS IN MOLECULAR BIOLOGY, Ausubel, ed. John Wiley & Sons, Inc.,New York (1997); LABORATORY TECHNIQUES IN BIOCHEMISTRY AND MOLECULARBIOLOGY: HYBRIDIZATION WITH NUCLEIC ACID PROBES, Part I. Theory andNucleic Acid Preparation, Tijssen, ed. Elsevier, N.Y. (1993).

Another useful means of obtaining and manipulating nucleic acids used inthe compositions and methods of the invention is to clone from genomicsamples, and, if necessary screen and re-clone inserts isolated (oramplified) from, e.g., genomic clones or cDNA clones or other sources ofcomplete genomic DNA. Sources of genomic nucleic acid used in themethods and compositions of the invention include genomic or cDNAlibraries contained in, or comprised entirely of, e.g., mammalianartificial chromosomes (see, e.g., Ascenzioni (1997) Cancer Lett. 118:135-142; U.S. Pat. Nos. 5,721,118; 6,025,155) (including humanartificial chromosomes, see, e.g., Warburton (1997) Nature 386:553-555;Roush (1997) Science 276:38-39; Rosenfeld (1997) Nat. Genet.15:333-335); yeast artificial chromosomes (YAC); bacterial artificialchromosomes (BAC); P1 artificial chromosomes (see, e.g., Woon (1998)Genomics 50:306-316; Boren (1996) Genome Res. 6:1123-1130); PACs (abacteriophage P1-derived vector, see, e.g., Ioannou (1994) Nature Genet.6:84-89; Reid (1997) Genomics 43:366-375; Nothwang (1997) Genomics41:370-378; Kern (1997) Biotechniques 23:120-124); cosmids, plasmids orcDNAs.

Amplification of Nucleic Acids

Amplification using oligonucleotide primers can be used to generatenucleic acids used in the compositions and methods of the invention, todetect or measure levels of test or control samples hybridized to anarray, and the like. The skilled artisan can select and design suitableoligonucleotide amplification primers. Amplification methods are alsowell known in the art, and include, e.g., polymerase chain reaction, PCR(PCR PROTOCOLS, A GUIDE TO METHODS AND APPLICATIONS, ed. Innis, AcademicPress, N.Y. (1990) and PCR STRATEGIES (1995), ed. Innis, Academic Press,Inc., N.Y., ligase chain reaction (LCR) (see, e.g., Wu (1989) Genomics4:560; Landegren (1988) Science 241:1077; Barringer (1990) Gene 89:117);transcription amplification (see, e.g., Kwoh (1989) Proc. Natl. Acad.Sci. USA 86:1173); and, self-sustained sequence replication (see, e.g.,Guatelli (1990) Proc. Natl. Acad. Sci. USA 87:1874); Q Beta replicaseamplification (see, e.g., Smith (1997) J. Clin. Microbiol.35:1477-1491), automated Q-beta replicase amplification assay (see,e.g., Burg (1996) Mol. Cell. Probes 10:257-271) and other RNA polymerasemediated techniques (e.g., NASBA, Cangene, Mississauga, Ontario); seealso Berger (1987) Methods Enzymol. 152:307-316; Sambrook; Ausubel; U.S.Pat. Nos. 4,683,195 and 4,683,202; Sooknanan (1995) Biotechnology13:563-564.

Polypeptides

The invention is directed to modified polypeptides and articles ofmanufacture comprising arrays with immobilized polypeptides, peptidesand peptidomimetics. The polypeptide is modified by reaction with acompound having the formula: R₁—X—R₂, where R₁ is a cyclic ether groupor an amino group, R₂ is an alkoxysilane group and X is a moietychemically suitable for linking the cyclic ether group or the aminogroup to the alkoxysilane group. As noted above, the terms“polypeptide,” “protein,” and “peptide,” used to practice the invention,include compositions of the invention that also include “analogs,” or“conservative variants” and “mimetics” or “peptidomimetics.” The terms“mimetic” and “peptidomimetic” refer to a synthetic chemical compounds.The mimetic can be either entirely composed of synthetic, non-naturalanalogues of amino acids, or, is a chimeric molecule of partly naturalpeptide amino acids and partly non-natural analogs of amino acids. Themimetic can also incorporate any amount of natural amino acidconservative substitutions as long as such substitutions also do notsubstantially alter the mimetics' structure and/or activity. Polypeptidemimetic compositions can contain any combination of non-naturalstructural components, which are typically from three structural groups:a) residue linkage groups other than the natural amide bond (“peptidebond”) linkages; b) non-natural residues in place of naturally occurringamino acid residues; or c) residues which induce secondary structuralmimicry, i.e., to induce or stabilize a secondary structure, e.g., abeta turn, gamma turn, beta sheet, alpha helix conformation, and thelike. A polypeptide can be characterized as a mimetic when all or someof its residues are joined by chemical means other than natural peptidebonds. Individual peptidomimetic residues can be joined by peptidebonds, other chemical bonds or coupling means, such as, e.g.,glutaraldehyde, N-hydroxysuccinimide esters, bifunctional maleimides,N,N′-dicyclohexylcarbodiimide (DCC) or N,N′-diisopropylcarbodiimide(DIC). Linking groups that can be an alternative to the traditionalamide bond (“peptide bond”) linkages include, e.g., ketomethylene (e.g.,—C(═O)—CH₂— for —C(═O)—NH—), aminomethylene (CH₂—NH), ethylene, olefin(CH═CH), ether (CH₂—O), thioether (CH₂—S), tetrazole (CN₄—), thiazole,retroamide, thioamide, or ester (see, e.g., Spatola (1983) in Chemistryand Biochemistry of Amino Acids, Peptides and Proteins, Vol. 7, pp267-357, “Peptide Backbone Modifications,” Marcell Dekker, NY). Apolypeptide can also be characterized as a mimetic by containing all orsome non-natural residues in place of naturally occurring amino acidresidues; non-natural residues are well described in the scientific andpatent literature. The skilled artisan will recognize that individualsynthetic residues and polypeptides incorporating mimetics can besynthesized using a variety of procedures and methodologies, which arewell described in the scientific and patent literature, e.g., OrganicSyntheses Collective Volumes, Gilman, et al., supra. Polypeptidesincorporating mimetics can also be made using solid phase syntheticprocedures, as described, e.g., by U.S. Pat. No. 5,422,426. Peptides andpeptide mimetics can also be synthesized using combinatorialmethodologies. Various techniques for generation of peptide andpeptidomimetic libraries are well known, and include, e.g., multipin,tea bag, and split-couple-mix techniques; see, e.g., al-Obeidi (1998)Mol. Biotechnol. 9:205-223; Hruby (1997) Curr. Opin. Chem. Biol.1:114-119; Ostergaard (1997) Mol. Divers. 3:17-27; Ostresh (1996)Methods Enzymol. 267:220-234. Modified polypeptide and peptides can befurther produced by chemical modification methods, see, e.g., Belousov(1997) Nucleic Acids Res. 25:3440-3444; Frenkel (1995) Free Radic. Biol.Med. 19:373-380; Blommers (1994) Biochemistry 33:7886-7896. Thesepeptides can also be synthesized, whole or in part, using chemicalmethods well known in the art (see e.g., Caruthers (1980) Nucleic AcidsRes. Symp. Ser. 215-223; Horn (1980) Nucleic Acids Res. Symp. Ser.225-232; Banga, A. K., Therapeutic Peptides and Proteins, Formulation,Processing and Delivery Systems (1995) Technomic Publishing Co.,Lancaster, Pa. Peptide synthesis can be performed using varioussolid-phase techniques (see e.g., Roberge (1995) Science 269:202;Merrifield (1997) Methods Enzymol. 289:3-13) and automated synthesis maybe used.

Arrays, or “BioChips”

The invention provides “arrays” or “microarrays” or “biochips” or “chip”comprising the modified biological molecules of the invention, includingthe modified nucleic acids and polypeptides of the invention. Arrays aregenerically a plurality of target elements immobilized onto the surfaceof the array as defined “clusters,” or “biosites,” each target elementcomprising a one or more biological molecules (e.g., nucleic acids orpolypeptides) immobilized a solid surface for association (e.g.,specific binding or hybridization) to a sample. The immobilized nucleicacids can contain sequences from specific messages (e.g., as cDNAlibraries) or genes (e.g., genomic libraries), including a human genome.Other target elements can contain reference sequences and the like. Thebiological molecules of the arrays may be arranged on the solid surfaceat different sizes and different densities. The densities of thebiological molecules in a cluster and the number of clusters on thearray will depend upon a number of factors, such as the nature of thelabel, the solid support, and the like. Each cluster/biosite maycomprise substantially the same biological molecule (e.g., nucleic acidor polypeptide), or, a mixture of biological molecules (e.g., nucleicacids of different lengths and/or sequences). Thus, for example, acluster/biosite may contain more than one copy of a cloned piece of DNA,and each copy may be broken into fragments of different lengths. Thesurface onto which the modified biological molecules of the inventionare immobilized can include nitrocellulose, glass, quartz, fused silica,plastics and the like, as discussed further, below. The compositions andmethods of the invention can incorporate in whole or in part designs ofarrays, and associated components and methods, as described, e.g., inU.S. Pat. Nos. 6,197,503; 6,174,684; 6.156,501; 6,093,370; 6,087,112;6,087,103; 6,087,102; 6,083,697; 6,080,585; 6,054,270; 6,048,695;6,045,996; 6,022,963; 6,013,440; 5,959,098; 5,856,174; 5,843,655;5,837,832; 5,770,456; 5,723,320; 5,700,637; 5,695,940; 5,556,752;5,143,854; see also, e.g., WO 99/51773; WO 99/09217; WO 97/46313; WO96/17958; WO 89/10977; see also, e.g., Johnston (1998) Curr. Biol.8:R171-R174; Schummer (1997) Biotechniques 23:1087-1092; Kern (1997)Biotechniques 23:120-124; Solinas-Toldo (1997) Genes, Chromosomes &Cancer 20:399-407; Bowtell (1999) Nature Genetics Supp. 21:25-32;Epstein (2000) Current Opinion in Biotech. 11:36-41; Mendoza (1999Biotechniques 27: 778-788; Lueking (1999) Anal. Biochem. 270:103-111;Davies (1999) Biotechniques 27:1258-1261.

Substrate Surfaces

The articles of manufacture of the invention comprising arrays can havesubstrate surfaces of a rigid, semi-rigid or flexible material. Thesubstrate surface can be flat or planar, be shaped as wells, raisedregions, etched trenches, pores, beads, filaments, or the like.Substrates can be of any material upon which a “capture probe” can bedirectly or indirectly bound. For example, suitable materials caninclude paper, glass (see, e.g., U.S. Pat. No. 5,843,767), ceramics,quartz or other crystalline substrates (e.g. gallium arsenide), metals,metalloids, polacryloylmorpholide, various plastics and plasticcopolymers, Nylon™, Teflon™, polyethylene, polypropylene,poly(4-methylbutene), polystyrene, polystyrene/latex, potymethacrylate,poly(ethylene terephthalate), rayon, nylon, poly(vinyl butyrate),polyvinylidene difluoride (PVDF) (see, e.g., U.S. Pat. No. 6,024,872),silicones (see, e.g., U.S. Pat. No. 6,096,817), polyformaldehyde (see,e.g., U.S. Pat. Nos. 4,355,153; 4.652,613), cellulose (see, e.g., U.S.Pat. No. 5,068,269), cellulose acetate (see, e.g., U.S. Pat. No.6,048,457), nitrocellulose, various membranes and gels (e.g., silicaaerogels, see, e.g., U.S. Pat. No. 5,795,557), paramagnetic orsuperparamagnetic microparticles (see, e.g., U.S. Pat. No. 5,939,261)and the like. Reactive functional groups can be, e.g., hydroxyl,carboxyl, amino groups or the like. Silane (e.g., mono- anddihydroxyalkylsilanes, aminoalkyltrialkoxysilanes,3-aminopropyl-triethoxysilane, 3-aminopropyltrimethoxysilane) canprovide a hydroxyl functional group for reaction with an aminefunctional group.

Generating Molecular Profiles of Sample Nucleic Acids

The invention provides compositions and methods for generating amolecular profile of a nucleic acid sample, such as a sample of genomicDNA or a cDNA library. The invention provides articles of manufactureand methods for contacting array-bound nucleic acids with a samplecontaining nucleic acids and detecting the binding of the sample nucleicacids to the array, thereby generating a molecular profile of the samplenucleic acid. In alternative aspects of the methods of the invention,the molecular profile can be a comparative genomic hybridization (CGH)reaction; detection of a genomic DNA amplification, a genomic DNAdeletion, or a genomic DNA insertion; detection of a point mutation,such as identification of a single-nucleotide polymorphism (SNP);differential methylation hybridization (DMH), where the array-boundnucleic acids are CpG island tags; detection of transcriptionally activeregions of a genome (using, e.g., nuclear run-off assays); analysis of achromatin structure; and analysis of a telomeric structure (such astelomeric erosion or telomeric addition). All of these procedures arewell known in the art, and any molecular biology procedure or analysis,can be performed using the modified biological molecules or arrays ofthe invention.

Comparative Genomic Hybridization (CGH)

In one aspect, the arrays and methods of the invention are used incomparative genomic hybridization (CGH) reactions. CGH is a molecularcytogenetics approach that can be used to detect regions in a genomeundergoing quantitative changes, i.e. gains or losses of copy numbers.Analysis of genomes of tumor cells can detect a region or regions ofanomaly under going gains and/or losses. Differential expression ofhundreds of genes can be analyzed using a cDNA array, thus facilitatingcharacterization of gene expression in normal and diseased tissues.Generating a molecular profile of a nucleic acid sample by comparativegenomic hybridization using methods and arrays of the invention can bepracticed with methods and compositions known in the art, see, e.g.,U.S. Pat. Nos. 6,197,501; 6,159,685; 5,976,790; 5,965,362; 5,856,097;5,830,645; 5,721,098; 5,665,549; 5,635,351; and. Diago (2001) AmericanJ. of Pathol. May;158(5):1623-1631; Theillet (2001) Bull. Cancer88:261-268; Werner (2001) Pharmacogenomics 2:25-36; Jain (2000)Pharmacogenomics 1:289-307.

Detection of Single-Nucleotide Polymorphisms (SNPs)

In one aspect, the arrays and methods of the invention are used todetect point mutations, such as single-nucleotide polymorphisms (SNPs).Arrays can be used for high-throughput genotyping approaches forpharmacogenomics, where numerous individuals are studied with thousandsof SNP markers. SNP mapping has accelerated complex disease genelocalization; detection of multiple SNPs associated with a disease in arelatively small linkage disequilibrium region can narrow the linkageregion for that disease, and, identification of susceptibility geneswill enable a better understanding of the mechanisms of the diseaseprocesses and will facilitate the discovery of new and more efficaciousmedicines. Generating a molecular profile of a nucleic acid sample bythe analysis and detection of SNPs using methods and arrays of theinvention can be practiced with methods and compositions known in theart, see, e.g., U.S. Pat. Nos. 6,221,592; 6,110,709; 6,074,831;6,015,888; and, Kwok (2000) Pharmacogenomics 1:95-100; Riley (2000)Pharmacogenomics 1:39-47; Kokoris (2000) Mol. Diagn. 5:329-340; Shi(2001) Clin. Chem. 47:164-172; Fan (2000) Genome Res. 10:853-860;Ianonne (2000) Cytometry 39:131-140; Cai (2000) Genomics.66:135-143;Chen (2000) Genome Res. 10:549-557; Syvanen (1999) Hum. Mutat. 13:1-10;Pastinen (1997) Genome Res. 7:606-614.

Differential Methylation Hybridization (DMH)

The arrays and methods of the invention are used in differentialmethylation hybridization (DMH), including, for example, CpG islandanalysis. In one aspect, the array-bound nucleic acids comprise CpGisland tags. In one aspect, the methods and arrays of the invention areused to identify, analyze and map hypermethylated or hypomethylatedregions of the genome. In one aspect, the sample nucleic acids cancomprise genomic DNA digested with at least one methylation-sensitiverestriction endonuclease and the molecular profile comprises detectionand mapping of hypermethylated (or hypomethylated) regions of thegenome. Any methylation-sensitive restriction endonuclease or equivalentendonuclease enzyme can be used, including, for example, NotI, SmaI,SacII, EagI, MspI, HpaI, Sau3AI and BssHII. In one aspect of the methodsof the invention, both a methylation-sensitive enzyme and itsmethylation insensitive isoschizomer is used; see, e.g., Robinson (2000)Chromosome Res. 8:635-643; described use of the methylation-sensitiveenzyme HpaII and its methylation insensitive isoschizomer MspI.Windhofer (2000) Curr. Genet. 37:194-199, described digestion of genomicDNA with the methylation-sensitive endonuclease Sau3AI and themethylation-insensitive endonuclease NdeII. See also, e.g., Muller(2001) J. Biol. Chem. 276:14271-14278; Memisoglu (2000) J. Bacteriol.182:2104-2112; Roth (2000) Biol. Chem. 381:269-272.

DNA methylation, or the covalent addition of a methyl group to cytosinewithin the context of the CpG dinucleotide, has profound effects on themammalian genome. These effects include transcriptional repression viainhibition of transcription factor binding or the recruitment ofmethyl-binding proteins and their associated chromatin remodelingfactors, X chromosome inactivation, imprinting and the suppression ofparasitic DNA sequences. DNA methylation is also essential for properembryonic development, DNA repair and genome stability. For example, DNAdemethylation influence on chromosome stability is modulated by asequence-specific chromatin structure (the invention also providesmodified biological molecules and arrays comprising chromatinstructures) (see, e.g., Vilain (2000) Cytogenet. Cell. Genet.90:93-101).

Normal methylation patterns are frequently disrupted in tumor cells withglobal hypomethylation accompanying region-specific hypermethylation.When these hypermethylation events occur within the promoter of a tumorsuppressor gene, they will silence the gene and provide the cell with agrowth advantage in a manner akin to deletions or mutations. Forexample, the Rb tumor suppressor pathway is frequently disrupted bymethylation-dependent silencing of the p16INK4A gene and stimulation ofRb degradation by a proteosomal subunit (see, e.g., Buendia (2000)Semin. Cancer Biol. 10:185-200). Reversal of abnormalities in DNAmethylation may therefore restore the tumor-suppressive function ofthese genes and provide a novel approach to cancer therapy (see, e.g.,Santini (2001) Ann. Intern. Med. 3;134(7):573-586. The transcriptionalsilencing of selected genes by DNA methylation plays a crucial role inthe development and progression of human gastrointestinal malignancies(see, e.g., Toyota (2000) J. Gastroenterol. 35:727-734). Generating amolecular profile of a nucleic acid sample by the analysis ofdifferential methylation and CpG islands using methods and arrays of theinvention can be practiced with methods and compositions known in theart, see, also, U.S. Pat. Nos. 6,214,556; 6,180,344; 5,851,762; and,WO0127317, WO9928498; WO0044934; and WO1999DE03747 19991119.

Analysis of Telomeric Structure

The arrays and methods of the invention are used in the analysis of atelomeric structure, such as telomeric erosion or telomeric addition.The maintenance of telomeres, which are specialized nucleoproteinstructures, is essential for chromosome stability. Without new synthesisof telomeres at chromosome ends the chromosomes shorten with progressivecell division. This eventually triggers either replicative senescence orapoptosis when telomere length becomes critically short. The regulationof telomerase activity in human cells plays a significant role in thedevelopment of cancer (telomerase is the enzyme that synthesizes thetelomere ends of linear eukaryotic chromosomes). Telomerase is tightlyrepressed in the vast majority of normal human somatic cells, butbecomes activated during cellular immortalization and in cancers. Thus,telomerase assays are useful for cancer detection and diagnosis (see,e.g., Hahn (2001) Ann Med 33:123-129; Meyerson (2000) J. Clin. Oncol.18:2626-2634; Meyerson (1998) Toxicol. Lett. 102-103:41-5). Using thearray-based telomeric structures of the invention will accelerateunderstanding of telomerase biology and lead to clinically relevanttelomerase-based therapies. Generating a molecular profile of a nucleicacid sample by the analysis of telomeric structures using methods andarrays of the invention can be practiced with methods and compositionsknown in the art, see, e.g., U.S. Pat. Nos. 6,221,590; 6,221,584;6,022,709; 6,007,989; 6,004,939; 5,972,605; 5,871,926; 5,834,193;5,830,644; 5,695,932; 5,645,986.

Analysis of Chromatin Structure

The arrays and methods of the invention are used in the analysis ofchromatin structure, including chromatin condensation, chromatindecondensation, histone phosphorylation, histone acylation, and the like(see, e.g., Guo (2000) Cancer Res. 60:5667-5672; Mahlknecht (2000) Mol.Med. 6:623-644). Chromatin structure remodeling occurs in certaincancers (see, e.g., Giamarchi (2000) Adv. Exp. Med. Biol. 480:155-161).Chromatin structure affects nuclear processes that utilize DNA as asubstrate, e.g., transcription, replication, DNA repair, and DNAorganization within the nucleus. Chromatin structure analysis is usefulin fertility assessment; for example, sperm with decondensed chromatinare infertile. DNA damage in patients with untreated cancer can bemeasured using a sperm chromatin structure assay (see, e.g., Kobayashi(2001) Fertil. Steril. 75:469-475). Generating a molecular profile of anucleic acid sample by the analysis of chromatin structure using themethods and arrays of the invention can be practiced with methods andcompositions known in the art, see, e.g., U.S. Pat. Nos. 6,204,064;6,187,749; 6,097,485; 5,972,608; 5,919,621; 5,470,709; and, Dreyer(2000) Anal. Cell Pathol. 20:141-150; Hong (2001) Acta Cytol.45:163-168; Evenson (1991) Reprod. Toxicol. 5:115-125.

Nuclear Run-Off Assay

The arrays and methods of the invention are used in the detection andanalysis of transcriptionally active regions of nucleic acid, e.g.,transcriptionally active regions of a genome. In one aspect, a sample ofnucleic acid can be derived from a nuclear run-off assay and detectedand analyzed by the compositions and methods of the invention. Inanother aspect, nuclear run-off samples are modified by the methods ofthe invention. In one aspect, these modified nucleic acids areimmobilized onto an array as set forth in the invention.

Generating a molecular profile of a nucleic acid sample by the detectionand analysis of transcriptionally active regions of nucleic acid by,e.g., nuclear run-off assays, using the methods and arrays of theinvention can be practiced with methods and compositions known in theart, see, e.g., U.S. Pat. Nos. 6,200,960; 6,184,032; 6,175,060;6,159,751; 6,022,694; 5,994,523; and, Delany (2001) Methods Mol. Biol.151:321-333; Srivastava (1998) Methods Mol. Biol. 86:201-207; Greene(1994) J. Biochem. Biophys. Methods 29:179-187; Srivastava (1994)Methods Mol. Biol. 31:281-288.

It will be readily apparent to one skilled in the art that varioussubstitutions and modifications may be made to the invention disclosedherein without departing from the scope and spirit of the invention. Itis understood that the examples and aspects described herein are forillustrative purposes only and that various modifications or changes inlight thereof will be suggested to persons skilled in the art and are tobe included within the spirit and purview of this application and scopeof the appended claims.

EXAMPLES

The following example is offered to illustrate, but not to limit theclaimed invention.

Example 1 Preparation of Modified Nucleic Acid Using3-glycidoxypropyltrimethoxysilane

The following example describes making and using one aspect of modifiednucleic acid of the present invention. The purpose of the chemicalmodification is to enable the nucleic acid to be readily affixed to anunderivatized solid surface. In this example, the nucleicacid—preferably DNA—is modified by reaction with3-glycidoxypropyltrimethoxysilane (GPTS), according to FIG. 1. GPTS hasin fact been previously used to derivatize a glass surface upon which(unmodified) DNA samples are then contacted and immobilized. Yet the useof GPTS is for the opposite purpose: to modify the DNA for subsequentattachment to an underivatized glass surface, has not been previouslydisclosed nor suggested. Moreover, GPTS—since it contains an epoxidegroup—is known to damage DNA in vivo. For these reasons, its use toderivatize DNA is actually discouraged by the prior art.

Schematically, affixing the nucleic acid to the solid support consistsessentially of two steps. In the first, the nucleic acid reacts with theepoxide end of the GPTS molecule; in the second step, the glass surfacereacts with the other end, or the silane end of the GPTS-modifiednucleic acid, thereby affixing the nucleic acid onto an underivatizedglass surface. The entire reaction is rapid, is characterized by afavorable equilibrium, and occurs under very mild conditions using aminimum of inexpensive reagents. Though there quite obviously arenumerous ways to carry out either step of the reaction, the preferredmethod is shown in this and the following example.

As depicted in FIG. 1, a chemical compound having a cyclic or ring etherand an alkoxysilane—in this instance ethylene oxide andtrimethyloxysilane, respectively—comprise the two ends of the compound;the two ends are connected by a four-carbon ether linkage. The compoundshown is 3-glycidoxypropyltrimethoxysilane or GPTS. In the first step,DNA is reacted with GPTS at basic pH, preferably above 9.5, to form themodified DNA. The modified DNA is then reacted with an underivatizedglass (or other silanol-containing) surface at neutral pH, thusimmobilizing the DNA onto the glass surface. In the first step, the ringether functionality reacts with the DNA. Again, the ring ether need notbe ethylene oxide, as it is in GPTS, although the small ring ispreferred to increase reactivity of the ether functionality, which isrelatively unreactive.

The first reaction, leading to the derivatized DNA, is a ring-openingreaction likely involving carbon 5 of the ribose ring of the DNA. Thisderivatized DNA is unusually stable and can be stored for long periodsof time prior to actual use. The second reaction, immobilizing thederivatized DNA onto the glass surface, is a simple substitutionreaction creating an Si—O—Si linkage in the glass surface, and removingone of the alkoxy groups from the GPTS molecule.

Example 2 Preparation of Modified Nucleic Acid Using3-aminopropyltriethoxysilane

The following example describes making and using another aspect ofmodified nucleic acids of the present invention. The purpose of thechemical modification is to enable the nucleic acid to be readilyaffixed to an underivatized solid surface. In this example, the nucleicacid, preferably DNA, is modified by reaction with3-aminopropyltrimethoxysilane, according to FIG. 2. As in example 1,affixing the nucleic acid to the solid support consists essentially oftwo steps. In the first, the nucleic acid reacts with the amino end ofthe 3-aminopropyltrimethoxysilane molecule; in the second step, theglass surface reacts with the other end, or the silane end of the3-aminopropyltrimethoxysilane-modified nucleic acid, thereby affixingthe nucleic acid onto an underivatized glass surface.

As in example 1, the entire reaction is rapid, is characterized by afavorable equilibrium, and occurs under very mild conditions using aminimum of inexpensive reagents. Though there quite obviously arenumerous ways to carry out either step of the reaction, the preferredmethod is shown in this and the following example.

As depicted in FIG. 2, a chemical compound having an amino group and analkoxysilane—in this instance—NH₂ and triethyloxysilane,respectively-comprise the two ends of the compound; the two ends areconnected by a propyl linkage. The compound shown is3-aminopropyltriethoxysilane. In the first step, DNA is reacted with3-aminopropyltriethoxysilane at neutral pH in the presence of sodiumbisulfite, or equivalent.

The first reaction, leading to the derivatized DNA, is transaminationreaction of the cytosine residues on nucleic acids. The second reaction,as in Example 1, involving immobilizing the derivatized DNA onto theglass surface, is a simple substitution reaction. It creates an Si—O—Silinkage in the glass surface and removes one of the alkoxy groups fromthe GPTS molecule.

Example 3 Preparation of a High Density, Microarray Using ModifiedNucleic Acid

The following example describes making a high-density microarray of theinvention.

Once the modified nucleic acids of the present invention, such as thosedescribed in Examples 1 and 2, are prepared, they can then be exploitedin a variety of ways, including, e.g., to make a high density array.Again, these modified nucleic acids (particularly DNA) can beimmobilized onto a glass surface simply by contacting the modified DNAonto the underivatized surface. The significance of this is, among otherthings, that spreading (migration of the DNA sought to be immobilizedfrom the desired location) and non-specific probe sticking (caused byderivatization of the glass surface which creates a net positiveelectrostatic charge upon the surface which attracts the net negativelycharged DNA) are essentially eliminated.

These advantages allow the creation of extraordinarily high-densitymicroarrays, which is highly desirable. For instance, due to theelimination of spreading, and the effective elimination of probesticking, a single small glass surface can contain virtually thousandsof DNA samples to be tested, each of which is microscopic in size, allimmobilized upon a single glass surface. Indeed, one can construct amicroarray consisting of multiple single sample spots smaller than 50microns placed upon a glass surface.

A high-density microarray consisting of multiple DNA samples of thistype is also easily constructed in accordance with the presentinvention. The modified DNA can be prepared (for instance, in accordancewith Examples 1 and 2) well in advance of actual use. These chemicallymodified DNA samples are analogous to “DNA chips” that can then bereadily “imprinted” upon an unaltered glass sheet in, for instance, gridfashion. FIG. 3 illustrates one aspect of a device for preparing such ahigh-density microarray using the DNA chips of the present invention. Inone preferred aspect, the device is made from a plurality of inexpensivecommercially available capillary micropipets, preferably 10 cmmicropipets, although other sizes will, of course, work. As depicted inFIG. 3 each 10 cm micropipet is pulled to make a taper at one end. Theyare arranged in a hexagonal close-packed array, bounded by a squareframe. The micropipets can be glued to one another to form a stable unitWithin the frame. The tapered ends (FIG. 3B) are cut off and polished tooptical flatness.

To prepare the microarray, the tips of the device are dipped into amulti-well container that contains the (chemically modified inaccordance with the present invention) DNA samples to be tested, andwhose wells are aligned with the micropipets of the device. Upon contactof the tips into the wells, a small portion of each DNA sample isdeposited into the micropipet corresponding to the particular well bysimple capillary action. The size of the spot can be carefullycontrolled by the size of the tapered end. Using this device and the DNAchips of the present invention, thousands of samples can be arrayed in anarrow area, simultaneously and without the need for expensive robotics.Indeed, the method (comprising the DNA chips and pipet device) of thepresent invention has been shown to be even more efficient than methodsusing high-speed spotting robots. Finally, the compounds, methods anddevices of the present invention are readily incorporated into apre-packaged kit for commercial sale.

The high-density microarray of the present invention can also be readilyincorporated into the microarray systems of the art, such as thosedisclosed in the art section above. For instance, fluorescent in situhybridization (FISH) and the method described in Shalon (1996) 6 GenomeRes. 639 (1996), describing a microarray system is presented foranalyzing DNA samples that involves making microarrays of DNA samples onglass substrates, probing them by hybridization with complexfluorescent-labeled probes, and using a laser-scanning microscope todetect the fluorescent signals representing hybridization. Similarly,Sargent, et al. (U.S. Pat. No. 5,601,982) discloses a method andapparatus for determining the sequence of polynucleotides involvingscanning the nucleic acids by scanning tunneling microscopy.

One skilled in the art recognizes that this invention is not limited tousing only nucleic acids. Other biological molecules, such asbiopolymers, e.g., DNA, RNA, proteins, peptides or polypeptides, andpolysaccharides, can be directly activated using the methods of theinvention, such as bifunctional silane compounds or other crosslinkingreagents, resulting in an immobilized biologic molecule, e.g., abiopolymer, to a solid surface. This invention demonstrates that thetarget molecules to be arrayed (i.e., immobilized) are first modified sothat they have gained a binding affinity for solid surfaces withoutlosing their probing (e.g., hybridization) abilities. Becausemodification is a separate process, virtually any biological moleculecan be modified and arrayed (immobilized). Thus, a skilled artisanrealizes that this invention is not limited to nucleic acids, but canalso be used for a spectrum of biological molecules.

Example 4 Preparation of Modified Nucleic Acids

The following example demonstrates methods for preparing modifiedbiological molecules of the invention by describing the modification ofnucleic acids.

Using Halogenated Silanes: This example describes another form ofmodified nucleic acid of the present Invention. Again, the purpose ofthe chemical modification disclosed and claimed here is to enable tonucleic acid to be readily affixed to an underivatized solid surface,e.g., ordinary quartz glass. According to FIG. 4, a modified nucleicacid in accordance with the present invention is prepared by reactingunmodified nucleic acid under near neutral pH with suitable silanecompounds. The “X” in FIG. 4 can refer to any halide, preferably Cl, Br,or I; R₁, R₂ and R₃, can be the same or different, including, —OCH₃, and—OC₂H₅. In particularly, preferred aspects, the halogenated silanedepicted to the left of the arrow in FIG. 4 is8-bromocytltrichlorosilane, 8-bromocytltrimethoxysilane,4-chlorobutylmethyldichlorosilane, and 3-iodopropyltrimethoxysilane.

The conversion depicted in FIG. 4 was performed as follows. Thehalogenated silane was dissolved in dimethylformamide (DMF) at aconcentration of about 30 mM. Next, 3 μg to 10 ∥g of nucleic acid wasdissolved in 100 ul of 0.01 M phosphate buffer (pH 7.0). Then 1 to 3 μgof 30 mM halogenated silane was added, the solution is then mixed well,and allowed to react at about 37° C. for about 3 hours (alternatively,it can be reacted at ambient temperature overnight). After reaction, thedesired product—the modified nucleic acid—is purified by ethanolprecipitation; then the modified nucleic acid is dissolved in water.

Example 5 Preparation of Arrays and Controlling Spot (Cluster) Densityand Size

The following example demonstrates an exemplary method for manufacturingthe arrays, or “biochips” of the invention.

As discussed throughout the present application, one particularadvantage of the present invention is that it allows the investigator toprepare unusually high-density microarrays to conduct nucleic acidstudies. This example is best understood in relation to example 3, whichdisclosed the preparation of a high-density microarray in accordancewith the present invention. This example discloses enhanced methods forcontrolling the size and density of the individual nucleic acid “spots”or “clusters” on the solid supports, in accordance with the presentinvention.

Small “spot” or “cluster” size, in relation to high-density microarrays,allows higher sample density (i.e., more samples per unit area) andsuperior detection sensitivity (because the signals are less diffuse).In the conventional solid support systems, the skilled artisan faces acrucial dilemma. An ordinary clean quartz glass surface—of the type usedin the experiments described here—is very hydrophilic. Thus, nucleicacid samples will naturally tend to spread out when placed on the glasssurface. Again, this is undesirable. To mitigate spreading, the skilledartisan can treat the surface to make it more hydrophobic—e.g., eitherpretreating the surface with a hydrophobic agent, or simply bydehydrating the surface. Naturally, either of these options makes theglass surface less reactive towards silane-modified nucleic acids.

In a family of aspects of the present invention discussed in thisexample, the skilled artisan is spared this dilemma. More specifically,spreading can be eliminated yet the reactivity of the surface towardsthe modified nucleic acids can be maintained through the use of anothertype of silanes of the present invention. For instance, one quitegeneral aspect of these silanes after hydrolysis contains an Si(OH)₃ ateach end, linked by a hydrophobic group. See FIG. 6. Any of a variety ofhydrophobic linkers can be used. Particularly preferred aspects include:1,6-Bis-trichlorosilyhexane, 1,8-Bis-trichlorosilyloctane,1,6-Bis-trimethoxysilyhexane, and 1,4 Bis-trimethoxysilylethylbenzene.Thus, according to these aspects of the present invention, one end ofthe silane attaches to the surface, and the other end remains reactiveto the modified biological molecule, e.g., nucleic acid. The hydrophobiclinker confers hydrophobicity to the surface. Thus, the skilled artisancan readily see how the electrostatic properties of the surface(hydrophobic versus hydrophilic) can be readily modulated—e.g., thechain length of the linker can be adjusted to control hydrophobicity,and the surface reactivity can be controlled by adjusting the amount ofsilane contacted with the surface.

To prepare the solid supports in accordance with this aspect of thepresent invention, the glass surface was cleaned by slowly boiling in 3M HCl for about 2 hrs in a fume hood. Next, the surfaces were rinsedwith deionized water then kept in 0.1 M HCl until ready for use. Whenready for use, the surfaces were rinsed with doubly distilled deionizedwater to remove any extant acid, then rinsed in absolute ethanol. Next,the surfaces were immediately transferred to an ethanol solutioncontaining 0.0005% to 0.002% of the bifunctional silanes of this aspectof the invention. The surfaces were then treated at room temperature forabout 48 hours. The surfaces were then rinsed with ethanol and airdried. Finally, the glass surfaces were stored in a dust-freeenvironment until ready for use.

Example 6 Preparation of Modified Nucleic Acids Using Amine-ContainingSilane Compounds

The following example describes another form of modified nucleic acid ofthe present invention. In this family of aspects, the modified nucleicacid is prepared by reacting pristine nucleic acids with anamine-containing silane. Heuristically, the derivatization of nucleicacid with amine-containing silanes is comprised of two steps: (1) thehalogenation (or bromination, as shown) of the nucleic acid (FIGS. 5 a,5 b); and (2) the derivatization of the halogenated nucleic acid (FIG. 5c); As depicted in FIG. 5 a, 5 b, the reaction can occur in the presenceof N-bromosuccinimide under mild pH conditions; varying either of thesereaction variables allows the skilled biochemist to control the reactionrate. Also as evidenced by FIGS. 5 a, 5 b, the reaction normally occursat the guanine or cytosine base depending upon the pH—i.e., neutral toslightly basic pH favors reaction at the guanine residue, more basic pHfavors reaction at the cytosine residue.

Slightly different reaction protocols are preferably used depending uponwhether the nucleic acid is DNA or RNA. For DNA, 5 μg of DNA wasdissolved in 100 μl of 0.1 M NaHCO₃, to reach a pH of about 9.5. Thissolution is kept on ice for about 5 minutes. Contemporaneously, a freshN-bromosuccinimide solution at concentration of about 10 mM was preparedand also chilled on ice. Next, 1 μl of the N-bromosuccinimide solutionis added to the DNA solution; the solution was then stirred vigorously(to vortex). The reaction was then allowed to proceed on ice for about15 minutes. Next, 10 μl of 0.5 M aminosilane solution at pH about 9.5 toabout 12, was added to the bromine-activated DNA solution; this newmixture was allowed to react at 65° C. for about 2 hours. Finally, thesilane-modified DNA was purified by methods well known in the art;preferably, it is purified by ethanol precipitation, or equivalentprocedures.

For RNA, a similar, though slightly different protocol was used: 5 μg ofRNA was dissolved in 100 μl of 0.1 M phosphate buffer, to reach a pH ofabout 7.5. This solution is kept on ice for about 5 minutes.Contemporaneously, a fresh N-bromosuccinimide solution at concentrationof about 10 mM was prepared and also chilled on ice. Next, 1 μl of theN-bromosuccinimide solution is added to the DNA solution; the solutionwas then stirred vigorously (to vortex). The reaction was then allowedto proceed on ice for about 15 minutes. Next, 10 μl of 1 M aminosilanesolution at pH about 8.0, was added to the bromine-activated DNAsolution; this new mixture was allowed to react at 45° C. for about 2hours. Finally, the silane-modified DNA was purified by methods wellknown in the art; preferably, it is purified by ethanol precipitation,or equivalent procedures.

In these aspects the following silanes are available for these reasons:

R can be —CH₃, or —C₂H₅;

R₁ can be H, —CH₃, —C₂H₅, —OCH₃, or —OC₂H₅;

R₂ can be H, —CH₃, —C₂H₅, —OCH₃, or —OC₂H₅;

Further any other amino silane compound after hydrolysis that takes thefollowing form is useful:

Example 7 Preparation of Modified Biological Molecules (Biopolymers)Using 3-glycidoxypropyltrimethoxysilane

This example describes methods to modify biological molecules, e.g.,nucleic acids, using bifunctional silane compounds.

The purpose of the chemical modification is to enable sample (thebiological molecule) to be readily affixed to an underivatized solidsurface. In this example, a biopolymer is modified by reaction with3-glycidoxypropyltrimethoxysilane (GPTS).

Schematically, affixing the biopolymer to a solid surface consistsessentially of two steps. In the first, the biopolymer reacts with theepoxide end of the GPTS molecule; in the second step, the glass surfacereacts with the other end, or the silane end of the GPTS-modifiedbiopolymer, thereby affixing the biopolymer onto an underivatizedsurface.

A skilled artisan recognizes that a variety of bifunctional crosslinkingreagents could be used in the present invention (see above).Crosslinking reagents and the conditions required for their use are wellknown in the art, thus, one skilled in the art would be able toextrapolate the information provided by this application and utilizespecific crosslinking reagents and conditions to obtain a specificmodified biopolymer.

Example 8 Preparation of Modified Small Molecules

This example describes methods to modify biological molecules which maynot be described as biopolymers. As described above, any biologicalmolecule can be incorporated into the compositions and methods of theinvention. These non-biopolymers are first crosslinked to epoxidesilane-activated biopolymers, e.g., biopolymers activated according toExample 7. The crosslinking of these non-biopolymers, which aretypically small molecules, increases the size and stability of themolecule. Once the non-biopolymer is crosslinked to an activatedbiopolymer, e.g., polyethylene glycol (PEG) or DNA, these crosslinkedmolecules can be immobilized on a solid surface by direct deposition andcuring under proper conditions.

Example 9 Silanization of Amine-Containing Biopolymers such as Proteinsand Polypeptides (e.g., Antibodies), Polysaccharides and Lipids

This example describes methods to modify amine-containing biologicalmolecules by silanization. Such biological molecules include peptidesand polypeptides (e.g., antibodies), lipids and polysaccharides, inaddition to nucleic acids comprising amine groups. Nucleic acidscomprising amine groups include, e.g., nucleotide compositionscontaining aminooxy moieties, as described in U.S. Pat. No. 6,127,533.

Biopolymers are effectively silanized and arrayed onto glass surfaces.Biopolymers are first treated with 2-iminothiolane (commonly known as“Traut's Reagent”) or N-succinimidyl S-acetylthioacetate (SATA) orsuccinimidyl acetylthiopropionate (SATP) to introduce an activesulfhydryl functional group. The activated biopolymers are silanized byreacting with the epoxide silane compound as described previously undermild conditions. A typical reaction is depicted in FIG. 7; this exampleuses SATP and an epoxide silane comprising —Si(OCH₃)₃.

Antibodies are silanized by various methods. One such method is to firstdissolve an antibody in 0.1 M sodium phosphate buffer (pH 7.3) with 50mM NaCl and 10 mM EDTA at a concentration of about 1 to 3 mg/ml. Then,add 5 μl of 100 mM SATA or SATP in a DMSO solution to 1 ml antibodysolution and react at room temperature (RT) overnight. Next, add 100 μMof 1 M hydroxylamine hydrochloride and react at RT for one hour. Afterthe RT activation, add 10 μM of 0.2 M 3-glycidoxypropyltrimethoxysilane(epoxide silane) and react at RT for about 5 hours. Upon completion ofall reactions, antibodies are purified by gel filtration on a SephadexG25 column, or equivalent. The modified antibody is fixed on a glasssurface by direct deposition.

All patents, publications mentioned in this specification are indicativeof the level of those skilled in the art to which the inventionpertains. All patents, publications herein are incorporated by referenceto the same extent as if each individual publication was specificallyand individually indicated to be incorporated by reference.

One skilled in the art will readily appreciate that the presentinvention is well adapted to carry out the objects and obtain the endsand advantages mentioned as well as those inherent therein. Thechemically modified nucleic acids, their attachment to solid support,along with the sequences, methods, procedures, assays, molecules,devices and specific compounds described herein are presentlyrepresentative of the preferred aspects are exemplary and are notintended as limitations on the scope of the invention. Changes thereinand other uses will occur to those skilled in the art which areencompassed within the spirit of the invention and are defined by thescope of the claims.

1. A modified biological molecule comprising a biological moleculemodified by reaction with a compound having the formula: R₁—X—R₂,wherein R₁ is a cyclic ether group or an amino group, R₂ is analkoxysilane group and X is a moiety chemically suitable for linking thecyclic ether group or the amino group to the alkoxysilane group.
 2. Themodified biological molecule of claim 1, wherein the cyclic ether is acompound comprising an epoxide group.
 3. The modified biologicalmolecule of claim 2, wherein the epoxide is ethylene oxide.
 4. Themodified biological molecule of claim 1, wherein the cyclic ether is anoxirane group.
 5. The modified biological molecule of claim 1, whereinthe cyclic ether is a compound comprising an aromatic hydrocarbonepoxide group.
 6. The modified biological molecule of claim 1, whereinthe R₁ group reacts with the biological molecule.
 7. The modifiedbiological molecule of claim 6, wherein the R₁ group is covalently boundto the biological molecule.
 8. The modified biological molecule of claim1, wherein the biological molecule comprises a polypeptide, a peptide ora peptidomimetic.
 9. The modified biological molecule of claim 1,wherein the biological molecule comprises a polysaccharide, or an analogor a mimetic thereof.
 10. The modified biological molecule of claim 1,wherein the biological molecule comprises a lipid, or an analog or amimetic thereof.
 11. The modified biological molecule of claim 1,wherein the biological a molecule comprises a small molecule.
 12. Themodified biological molecule of claim 1, wherein the biological moleculecomprises a nucleic acid or an analog or mimetic thereof.
 13. Themodified biological molecule of claim 12, wherein the nucleic acidcomprises a DNA or an RNA.
 14. The modified biological molecule of claim12, wherein the nucleic acid reacts with the R₁ group at its 5′ end. 15.The modified biological molecule of claim 12, wherein the nucleic acidis an oligonucleotide.
 16. The modified biological molecule of claim 12,wherein the nucleic acid comprises a telomeric structure.
 17. Themodified biological molecule of claim 12, wherein the nucleic acidcomprises a chromatin structure.
 18. The modified biological molecule ofclaim 1, wherein cyclic ether is an epoxide group and the alkoxysilaneis —Si(OCH₃)₃, —Si(OC₂H₅)₃, —Si(OCH₃)H₂, —Si(OCH₃)(CH₃)₂, or—Si(OCH)₃)₂CH₃.
 19. The modified biological molecule of claim 1, whereincyclic ether is an epoxide group and the compound is3-glycidoxypropyltrimethoxysilane (GPTS).
 20. The modified biologicalmolecule of claim 1, wherein the R₁ amino group comprises a primaryamino group.
 21. The modified biological molecule of claim 1, wherein R₁is an amino group and the alkoxysilane is selected from the groupconsisting of —Si(OCH₃)₃, —Si(OC₂H₅)₃ and

wherein R₁, R₂ and R₃ are selected from the group consisting of —H,—CH₃, —OCH₃, and —OC₂H₃, and provided that at least one of R₁, R₂ or R₃is either —OCH₃ or —OC₂H₃.
 22. The modified biological molecule of claim1 wherein R₁ is an amino group and the compound is3-aminopropyltriethoxysilane. 23-81. (canceled)
 82. A method for makinga modified biological molecule comprising (a) providing a biologicalmolecule; (b) providing a compound having the formula: R₁—X—R₂ whereinR₁ is a cyclic ether group or an amino group, R₂ is an alkoxysilanegroup and X is a moiety chemically suitable for linking the cyclic ethergroup or the amino group to the alkoxysilane group; and (c) reacting thebiological molecule with the compound, thereby modifying the biologicalmolecule with the compound.
 83. (canceled)
 84. The method of claim 82further comprising selecting the biological molecule from a polypeptide,a peptide or a peptidomimetic.
 85. The method of claim 82 furthercomprising selecting the biological molecule from a polysaccharide, oran analog or a mimetic thereof.
 86. The method of claim 82 furthercomprising selecting the biological molecule from a lipid, or an analogor a mimetic thereof.
 87. The method of claim 82 further comprisingselecting the biological molecule comprises a nucleic acid or an analogor mimetic thereof.